Fibroblast activation protein alpha cleavable propeptides and methods of use

Abstract

Provided are propeptides cleavable by fibroblast activation protein alpha (FAPα), which find use in treating conditions associated with FAPα expression. The FAPα-cleavable propeptides also find use in imaging the location of FAPα activity in vivo. In some embodiments, the propeptides of the present disclosure comprise a membrane-interacting domain, a masking domain, and a FAPα cleavage site. The masking domain, when linked to the membrane-interacting domain, is effective to inhibit the interaction of the membrane-interacting domain with a phospholipid bilayer. The FAPα cleavage site is disposed between the membrane-interacting domain and the masking domain. The membrane-interacting domain can be conjugated to one or more therapeutic agents, non-limiting examples of which include radioisotopes. Also provided are methods of treating a condition associated with FAPα expression in a subject in need thereof, comprising administering to the subject an effective amount of a propeptide of the present disclosure.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 401,008, filed August 25, 2022, which is incorporated herein by reference in its entirety.

[0002] Statement of Government Support This invention was made with government support under R01 CA258297 and R01 AI161027 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING The Sequence Listing is provided herewith as Sequence Listing XML "UCSF-680WO_SEQ_LIST", created on August 24, 2023, and having a size of 48,677 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0004] Introduction Lutetium Lu177 dotatate (Lutathera®), iobenguane I131 (Azedra®), and 177 The recent FDA approval of Lu-PSMA617 (Pluvicto®), along with the emergence of promising experimental agents in clinical trials, highlights the burgeoning enthusiasm for investigating targeted radiation therapy (TRT) as a treatment modality for cancer. However, clinical experience with TRT indicates that it is rarely curative, and tumor responses are typically transient and / or variable among patients. Therefore, new strategies are needed to maximize the therapeutic benefit of TRT for cancer patients.

[0005] Within the past decade, the field of nuclear medicine has prioritized the development of low-MW radioligand therapy (RLT), which rapidly exits the bloodstream to minimize host toxicity yet remains an effective antitumor agent by targeting highly overexpressed cancer proteins. This transition was motivated by 30 years of clinical experience with various high-MW radioligands, such as immunoglobulins, which demonstrated that their long serum half-lives (3–7 days) resulted in radiation exposure to sensitive normal tissue compartments (e.g., bone marrow), narrowing or eliminating their therapeutic index. The evolution of radioligands targeting prostate-specific membrane antigen (PSMA) stands out as an instructive case study on the impact of radioligand pharmacokinetics (PK) on therapeutic index. 177 While various radiolabeled forms of IgG J591, including Lu-J591, have stalled in clinical trials due to dose-limiting toxicity, Pluvicto® (Lu-J591), a low MW radioligand with weaker affinity for PSMA and lower tumor uptake compared to J591, has been shown to be effective against IgG J591. 177 Lu-PSMA617) nevertheless achieved FDA approval for the treatment of prostate cancer in 2022, largely due to its more favorable safety profile.

[0006] However, treating cancer with low-MW RLT is challenging for many reasons. First, to deliver a sufficient dose to the tumor, the field is limited to only a few highly overexpressed proteins in cancer that can extract enough RLT from the circulation as the drug rapidly exits the body. Indeed, prominent RLT drug targets such as PSMA, somatostatin receptor type 2, carbonic anhydrase 9, and bombesin receptor are all very highly overexpressed on cancer cells (at least 10 per cell). 5 Second, the ligand / receptor complex is inherently unstable in biology and dissociates or degrades after endocytosis. Indeed, longitudinal PET studies in patients have shown that RLT is typically cleared from tumors within 96 hours, and in some extreme cases (e.g., FAPI PET), the radioisotope may leave the tumor within hours.

[0007] Fibroblast activation protein-α (FAPα) is a type II integral serine protease expressed by activated fibroblasts. Cancer-associated fibroblasts (CAFs) in the tumor stroma exhibit abundant and stable expression of FAPα, which plays an important role in promoting tumor growth, invasion, metastasis, and immunosuppression. The protein encoded by the human FAPα gene is a 760-amino acid, single-pass type II transmembrane protein consisting of a short cytoplasmic N-terminal portion (6 amino acids), a transmembrane region (7–26 amino acids), and a large extracellular domain. FAP is enzymatically active as a homodimer. It exhibits both post-proline dipeptidyl peptidase and endopeptidase activities, both of which depend on the catalytic triad containing Ser624, Asp702, and His734 in human and mouse FAPα. Due to the unique structure of proline, most proteases do not cleave the peptide bond adjacent to it. In some cases, the presence of proline therefore acts as a mechanism to prevent protein degradation or cleavage.

[0008] Normal tissues have low and generally undetectable levels of FAPα expression. However, FAPα is overexpressed in many tumor tissues, including breast cancer, colorectal cancer, pancreatic cancer, lung cancer, brain cancer, intrahepatic cholangiocarcinoma, and ovarian cancer. In addition, high levels of FAPα expression can be detected in some tumors derived from non-epithelial tissues, such as melanoma and myeloma. In these tumors, FAPα overexpression is often observed in the stroma, leading to its recognition as a universal marker for CAFs, although FAPα can also be detected in gastric cancer, pancreatic cancer, and melanoma cells. Details regarding FAPα expression in human malignant tumors can be found, for example, in Busek et al. (2018) Frontiers in Bioscience, Landmark 23:1933-1968. Summary of the Invention

[0009] Provided are propeptides cleavable by fibroblast activation protein alpha (FAPα), which find use in treating conditions associated with FAPα expression. The FAPα-cleavable propeptides also find use in imaging the location of FAPα expression in vivo. In some embodiments, the propeptides of the present disclosure comprise a membrane-interacting domain, a masking domain, and a FAPα cleavage site. The masking domain, when linked to the membrane-interacting domain, is effective to inhibit the interaction of the membrane-interacting domain with a phospholipid bilayer. The FAPα cleavage site is disposed between the membrane-interacting domain and the masking domain. The membrane-interacting domain may be conjugated to one or more therapeutic agents, non-limiting examples of which include radioisotopes. Also provided are methods of treating a condition associated with FAPα expression in a subject in need thereof, comprising administering to the subject an effective amount of a propeptide of the present disclosure. [Brief explanation of the drawings]

[0010] [Figure 1] Top: Schematic diagram of a propeptide (sometimes referred to herein as a "restricted interacting peptide" or "RIP") according to an embodiment of the present disclosure. RIPs are low-MW (approximately 4 kDa) peptides that contain three domains, from N- to C-terminus: (i) a membrane-interacting domain (in this example, a membrane-binding antimicrobial peptide (AMP)) coupled to a payload (e.g., a chelator bound to a radioisotope), (ii) an endoprotease cleavage site, e.g., spanning P4-P4', and (iii) a peptide "masking domain" that prevents the AMP from adopting the helical conformation required for membrane binding. Bottom: In this example, upon cleavage of the propeptide by a target endoprotease in vivo, radiolabeled AMP is liberated, spontaneously adopts a helical conformation, and binds to nearby phospholipid membranes. [Figure 2]Schema illustrating the proposed benefits of RIP-based targeted radiation therapy (TRT). On the left, typical blood and tumor activity curves for large molecular weight targeted radiation therapy (e.g., immunoglobulins) are shown. High-MW targeted radiation therapy has high tumor uptake but dose-limiting toxicity due to slow clearance from the blood. Low-MW targeted radiation therapy (e.g., small molecule radioligand therapy) benefits from improved safety due to rapid serum clearance, but inherently rapid clearance limits tumor exposure. RIP achieves both desirable safety due to rapid clearance and high tumor AUC due to reproducible catalytic amplification of isotope accumulation in the tumor and persistent binding of the membrane-interacting peptide to the cell membrane. [Figure 3] Discovery of unique candidate P4-P4' fibroblast activation protein alpha (FAPα) substrate sequences through multi-substrate profiling by mass spectrometry (MSP-MS). Top: Icelogo depicting cumulative amino acid preferences for recombinant human FAPα. Bottom: Heatmap summarizing the most frequently incorporated amino acids at each site in the cleavage products. Brighter indicates enriched amino acids. X = no preferred amino acid. [Figure 4A-C] Kinetic data for three of the top P4-P4' 8mer peptides emerging from the MSP-MS assay. Kinetic properties were determined using FRET donor / acceptor substrate adducts and human recombinant FAP alpha. [Figure 5] Schematic diagram of a propeptide (SEQ ID NO: 53) according to an embodiment of the present disclosure. In this non-limiting example (sometimes referred to herein as "FRIP2"), from N-terminus to C-terminus, is (1) a Cu-labeled membrane interaction / binding domain, (2) a FAPα cleavage site, and (3) a masking domain. [Figure 6A-B]Representative coronal PET / CT images from male nu / nu mice bearing subcutaneous U87 tumors show that 64Cu-FRIP2 has higher tumor uptake compared to FRIP1, FRIP3, and two negative control RIPs, PAR1 (cleaved by thrombin) and GRIP B (cleaved by granzyme B). Images were acquired 24 hours after injection. On the right, mean tumor SUV values ​​for each mouse cohort are shown. *P<0.01. [Figure 7] Evaluation of 64Cu-FRIP2 uptake in U87 tumors compared to tumor models lacking FAPα. The data demonstrate the specificity of FRIP2 for FAPα. [Figure 8] Biodistribution data collected 24 hours after injection of 64Cu-FRIP2 in male nu / nu mice bearing subcutaneous U87 MG xenografts show high uptake in tumors compared with normal tissues. The normal tissues with the highest uptake were the liver and kidney. [Figure 9A-B] Data showing superior tumor uptake of a labeled propeptide of the present disclosure (64Cu-FRIP2 in this example) compared to FAPI-46. [Figure 10A-B] Longitudinal PET / CT study showing the uptake of 64Cu-FRIP2 and 64Cu-FAPI 46 in PC3-PIP tumors, a model with low FAP alpha expression. ROI analysis of tumor uptake (Figure 10A) shows that 64Cu-FRIP2 was significantly higher in the tumor compared to 64Cu-FAPI 46. Figure 10B shows representative cross-sectional images showing tumor uptake of either radiotracer over time. Orange arrows indicate the location of the tumor. [Figure 11] Schematic illustrating the advantages of a RIP-based approach for targeted radiation therapy (TRT). [Figure 12A-C]67Cu-FRIP2 Antitumor Evaluation Data. Figure 12A: Relative change in tumor volume from a cohort of male nu / nu mice bearing subcutaneous U87 xenografts treated with either vehicle or 67Cu-FRIP2. On day 0 of the study, mice (n=8 / arm) were treated with a single IV bolus of 67Cu-FRIP2 (1 mCi / mouse). Figures 12B-12C: IHC images of FAP alpha staining of U87 xenografts. Staining of HEK-FAP tumors (HEK293 stably overexpressing human FAP alpha) is shown to correlate with the level of staining in U87. [Figure 13A-B] Additional 67Cu-FRIP2 antitumor evaluation data. Figure 13A: Survival curves for each cohort. Endpoints were (1) tumor volume exceeding 2000 mm or (2) weight loss exceeding 20%. Figure 13B: Relative change in body weight for mice within a cohort. [Figure 14] Spider plot showing the change in individual tumor volume for the represented mice. [Figure 15A-B] Spider plots of tumor volumes from an antitumor evaluation study in male nu / nu mice bearing subcutaneous U87 tumors treated with vehicle, 67Cu-FAPI 46, or 67Cu-FRIP2 (Figure 15A). Mice received a single IV dose of 1 mCi of radioactivity on day 0 of the study. Figure 15B: Relative tumor volumes from each cohort (n=8 mice / arm) on day 13, the latest day on which all mice in the study were still viable. 67Cu-FRIP2 suppresses tumor growth more potently than 67Cu-FAPI 46. *P<0.01. [Figure 16] Survival curves for each cohort are shown in Figure 15. Endpoints were (1) tumor volume greater than 2000 mm3 or (2) weight loss greater than 20%. [Figure 17A-B]Radioligand therapy with 67Cu-FRIP2 in PC3-PIP. Figure 17A: 69-day antitumor evaluation data showing the effect of 67Cu-FRIP2 treatment on PC3-PIP subcutaneous xenografts (P<0.01). On day 0, mice were treated with vehicle (N=6) or 1 mCi of 67Cu-FRIP2 (N=9). Figure 17B: Survival data plotted on a Kaplan-Meier curve show significantly prolonged survival (P<0.001) in the treated arm compared to the arm receiving vehicle. [Figure 18] Mouse weight changes in the two cohorts treated with vehicle or 1 mCi of 67Cu-FRIP2 on day 0. No unsafe weight changes were observed in the treated arms. [Figure 19A-B] Radioligand therapy with 64Cu-FRIP2 in U87 MG. Figure 19A: 27-day antitumor evaluation data showing the effect of 64Cu-FRIP2 treatment on U87 MG subcutaneous xenografts (P<0.01). Mice were treated with vehicle (N=8) or 1.5 mCi of 64Cu-FRIP2 (N=8) on days 0 and 8. Figure 19B: Survival data plotted on Kaplan-Meier curves show significantly prolonged survival (P<0.01) in the treated arm compared with the arm receiving vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0011] Before describing the propeptides and methods of the present disclosure in more detail, it is to be understood that the propeptides and methods are not limited to particular embodiments described, as such may, of course, vary. Also, the scope of the propeptides and methods will be limited only by the appended claims, and therefore it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0012] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the Propeptides and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the Propeptides and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the Propeptides and methods.

[0013] Certain ranges are presented herein with numerical values ​​preceded by the term "about." As used herein, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number preceded by the term. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the propeptides and methods belong. Although any propeptides and methods similar or equivalent to those described herein can also be used in the practice or testing of the propeptides and methods, representative exemplary propeptides and methods are described here.

[0015] All publications and patents cited herein are incorporated by reference to disclose and describe the materials and / or methods in connection with which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present propeptides and methods are not entitled to antedate such publication, which dates may be different from the actual publication dates that may need to be independently confirmed.

[0016] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate to the use of such exclusive terminology as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0017] For clarity, it is understood that certain features of the propeptides and methods that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the propeptides and methods that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure, and to the extent such combinations encompass operable processes and / or compositions, each and every combination is disclosed herein as if it were individually and explicitly disclosed. In addition, all subcombinations listed in the embodiments describing such variables are also specifically encompassed by the present propeptides and methods, and each and every such subcombination is disclosed herein as if it were individually and explicitly disclosed herein.

[0018] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0019] Propeptide The present disclosure provides a propeptide cleavable by FAPα. According to some embodiments, the propeptide comprises a membrane-interacting domain and a masking domain that, when linked to the membrane-interacting domain, is effective to inhibit interaction of the membrane-interacting domain with a phospholipid bilayer. Such a propeptide further comprises a FAPα cleavage site located between the membrane-interacting domain and the masking domain.

[0020] A schematic diagram of a propeptide (sometimes referred to herein as a "restricted interacting peptide" or "RIP") according to embodiments of the present disclosure is provided in Figure 1. A RIP can be a low-MW (approximately 4 kDa) peptide consisting of three domains, from N- to C-terminus: (i) a membrane-interacting domain (in this example, a membrane-binding antimicrobial peptide (AMP)) coupled to a payload (e.g., a chelator bound to a radioisotope), (ii) an endoprotease cleavage site, e.g., spanning P4-P4', and (iii) a peptide "masking domain" that prevents the AMP from adopting the helical conformation required for membrane binding. Bottom: In this example, upon cleavage of the propeptide by the target endoprotease in vivo, the radiolabeled AMP is liberated, spontaneously adopts a helical conformation, and immediately binds to nearby phospholipid membranes.

[0021] The propeptides of the present disclosure find use in a variety of contexts. For example, when the membrane-interacting domain is stably associated (e.g., conjugated) with one or more therapeutic agents (e.g., radioisotopes), the propeptides find use in treating conditions associated with FAPα expression in a subject in need thereof. Non-limiting examples of such conditions include cancer. Figure 2 shows a schema illustrating the benefits of RIP-based targeted radiotherapy (TRT). On the left, typical blood and tumor activity curves for large molecular weight targeted radiotherapy (e.g., immunoglobulins) are shown. High-MW targeted radiotherapy has high tumor uptake but dose-limiting toxicity due to slow clearance from the blood. Low-MW targeted radiotherapy (e.g., small molecule radioligand therapy) benefits from improved safety due to rapid serum clearance, but inherently rapid clearance limits tumor exposure. RIPs achieve both desirable safety due to rapid clearance and high tumor AUC due to reproducible catalytic amplification of isotope accumulation in tumors and persistent binding of the membrane-interacting peptide to the cell membrane.

[0022] Also, for example, when the membrane interaction domain is detectably labeled (e.g., with a radioisotope), the propeptide finds use in assessing FAPα activity in a subject, for example, by in vivo imaging of cells labeled with the detectably labeled membrane interaction domain. As shown in the experimental section herein, the propeptide of the present disclosure surprisingly exhibits superior biodistribution compared to active site-directed FAPα radioligands currently undergoing clinical trials. Details regarding the propeptide of the present disclosure are now described.

[0023] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to designate a series of amino acid residues connected to one another by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 "standard" genetically encodable amino acids, amino acid analogs, or combinations thereof. The series of amino acid residues may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and peptides with modified peptide backbones.

[0024] As used herein, the term "propeptide" refers to a peptide whose activity is limited because the individual portions of the peptide are linked together, thus limiting or restricting the activity they may have when not linked to one another. The activity of the individual portions of the propeptide is released upon cleavage or destruction of one or more of the bonds holding the individual portions together.

[0025] According to some embodiments, propeptides of the present disclosure comprise between 10 and 50 amino acids. For example, in some embodiments, propeptides of the present disclosure comprise at least 10 amino acids, but no more than 50, no more than 45, no more than 40, no more than 35, or no more than 30 amino acids.

[0026] In certain embodiments, a propeptide of the present disclosure comprises, from N-terminus to C-terminus, a membrane interaction domain, a FAPα cleavage site, and a masking domain. As used herein in the context of a polypeptide's structure, "N-terminus" and "C-terminus" refer to the amino- and carboxyl-terminal ends of the polypeptide, respectively, while "towards the N-terminus" and "towards the C-terminus" refer to the relative positions of the amino acid sequence of the polypeptide toward the N-terminus and C-terminus, respectively, and may include residues at the N-terminus and C-terminus, respectively. "Adjacent N-terminus" or "adjacent C-terminus" refers to the position of a first amino acid residue relative to a second amino acid residue, where the first and second amino acid residues are covalently linked to provide a contiguous amino acid sequence.

[0027] Membrane interaction domain In certain embodiments, the membrane-interacting domain (sometimes referred to herein as a "membrane-binding domain") comprises multiple non-polar, hydrophobic amino acid residues and comprises an alpha-helical structure that, when unconstrained by the masking domain, can interact with a phospholipid bilayer, such as a cell membrane. Such secondary structure may appear before, during, or after insertion of the membrane-interacting domain into a phospholipid bilayer. The composition of the membrane-interacting peptides described herein is not strictly limited to non-polar, hydrophobic amino acid residues; thus, the peptides may comprise different types of amino acid residues, such as polar, uncharged, polar, basic, or polar, acidic amino acid residues.

[0028] Thus, in some cases, the membrane-interacting domain, when separated from the masking domain, contains an alpha-helical structure capable of inserting into a phospholipid bilayer. The alpha-helix is ​​a common motif in protein secondary structure and generally comprises a right-handed coiled or helical conformation stabilized by hydrogen bonds, in which the NH group of the first amino acid residue forms a hydrogen bond with the C=O group of an amino acid residue located four residues apart in the polypeptide chain. A typical alpha-helix contains approximately 3.6 amino acid residues per turn, forming a tightly packed structure. The side chains of the amino acid residues that make up the alpha-helix face outward. Different amino acid sequences have different propensities to form alpha-helices, due in part to the different chemical properties of the amino acid side chains.

[0029] According to some embodiments, the membrane interacting domain comprises about 5 to about 30 amino acid residues. For example, in some embodiments, the membrane interacting domain comprises at least 5 amino acids, but no more than 30, no more than 25, no more than 20, or no more than 15 amino acid residues.

[0030] In certain embodiments, the membrane-interacting domain comprises an antimicrobial peptide (AMP) or a portion thereof. AMPs or portions thereof may be incorporated into the propeptides of the present disclosure in their naturally occurring form, or may be modified to alter their chemical properties and adapt them for desired applications. For example, the membrane-interacting ability of an antimicrobial peptide can be strengthened or weakened by, for example, adding, removing, or substituting specific amino acid residues in the protein sequence. Such additions, removals, or substitutions can be made, for example, to introduce charged amino acid residues, to eliminate charged amino acid residues, to introduce hydrophobic amino acid residues, to remove hydrophobic amino acid residues, etc.

[0031] According to some embodiments, the antimicrobial peptide sequence can be altered by chemically modifying the peptide with disulfide bonds or other chemical modifications (e.g., amidation). Many antimicrobial peptides are naturally produced with such modifications to improve their efficacy in interacting with phospholipid membranes and their resistance to proteolysis.

[0032] In some embodiments, the membrane interaction domain comprises a protein from the temporin family. Proteins in the temporin family generally range in length from about 10 to about 14 amino acids. The consensus sequence for the temporin family of proteins, showing the most abundant amino acid found at each position, is FLP(I / L)IASLL(S / G)KLL (SEQ ID NO: 1). The consensus sequence for the temporin family of proteins, showing the common amino acid types found at each position, is X a X b X c X d X e X f Y a X g X h Y b Y * X i X j wherein X a , X b , X c , Xd , X e , X f , X g , X h , X i , and X j is a hydrophobic amino acid residue, and Y a and Y b is a hydrophilic amino acid residue, and Y * are charged amino acid residues. The following table shows the amino acid sequences of some temporin and temporin-like peptides that are useful in the propeptides and methods of the present disclosure.

[0033] As described above, the antimicrobial peptide sequence may be altered by removing or substituting one or more of the amino acid residues. For example, in some embodiments, the membrane-interacting domain comprises temporin-L, whose amino acid sequence is FVQWFSKFLGRIL (SEQ ID NO: 2). In other embodiments, the membrane-interacting domain comprises a derivative of temporin-L, whose amino acid sequence is FVQWFSKFLGKLL (SEQ ID NO: 3), in which the amino acid residues R and I at positions 11 and 12 of the temporin-L sequence are replaced with amino acid residues K and L, respectively. [Table 1]

[0034] In some embodiments of the present disclosure, the membrane interacting domain comprises a temporin or temporin-like peptide listed in Table 1, or a conservative amino acid substitution thereof. In some embodiments of the present disclosure, the membrane interacting domain comprises the sequence of temporin-L (FVQWFSKFLGRIL; SEQ ID NO: 2), or a conservative amino acid variant thereof.

[0035] In some embodiments of the present disclosure, the membrane interaction domain comprises protonectin having the amino acid sequence ILGTILGLLKGL (SEQ ID NO: 33), or a conservative amino acid variant thereof.

[0036] In some embodiments, the membrane-interacting domain may comprise a japonisin or japonisin-like peptide. In some embodiments of the present disclosure, the membrane-interacting peptide comprises the sequence of japonisin-1 (FFPIGVFCKIFKTC; SEQ ID NO: 34), or a conservative amino acid variant thereof. Japonisin can be naturally obtained from the skin of the Japanese brown frog (Rana japonica) and ranges in length from about 14 to about 21 amino acid residues.

[0037] Masking Domain As summarized above, the propeptides of the present disclosure include a masking domain that, when linked to a membrane-interacting domain, is effective to inhibit the interaction of the membrane-interacting domain with a phospholipid bilayer.

[0038] In certain embodiments, the masking domain comprises between 5 and 30 amino acids. For example, in some embodiments, the masking domain comprises at least 5 amino acids, but no more than 30, no more than 25, no more than 20, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, or no more than 10 amino acid residues.

[0039] According to some embodiments, the masking domain comprises a peptide derived from protease-activated receptor-1 (PAR-1). "Derived from," in the context of an amino acid sequence or polynucleotide sequence, is meant to indicate that the polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid, and is not meant to be limiting with respect to the source or manner in which the protein or nucleic acid is made.

[0040] In certain embodiments, when the masking domain comprises a peptide derived from PAR-1, the masking domain comprises the amino acid sequence QDPNDQYEPF (SEQ ID NO: 35). In other embodiments, when the masking domain comprises a peptide derived from PAR-1, the masking domain comprises the amino acid sequence RNPNDKYEPF (SEQ ID NO: 36).

[0041] FAPα cleavage site As summarized above, the propeptides of the present disclosure comprise a FAP alpha cleavage site located between the membrane interaction domain and the masking domain. According to some embodiments, the FAP alpha cleavage site comprises the amino acid sequence YHGPLAHX (SEQ ID NO: 37), HIGPTAAY (SEQ ID NO: 38), XXIPTNIR (SEQ ID NO: 39), HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), HYGPTVNK (SEQ ID NO: 42), XXWPMGMY (SEQ ID NO: 43), XXFPNMWS (SEQ ID NO: 44), XXGSQVFS (SEQ ID NO: 45), XXMPEEVA (SEQ ID NO: 46), or XXHPTKSF (SEQ ID NO: 47), where X is any amino acid.

[0042] In certain embodiments, the FAPα cleavage site comprises the FAPα-specific motif GP at positions P2-P1. For example, FAPα cleavage sites that can be incorporated into the propeptides of the present disclosure include, but are not limited to, FAPα cleavage sites comprising the sequence HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), or HYGPTVNK (SEQ ID NO: 42). According to some embodiments, the FAPα cleavage site comprises the sequence HQGPFWML (SEQ ID NO: 40). As shown in the experimental section herein and in Figure 4, the kcat / Km for the FAPα cleavage site comprising the sequence HQGPFWML (SEQ ID NO: 40) is approximately 80,000 M, the highest turnover number reported to date for an FAPα substrate. -1 seconds -1 It was found that:

[0043] therapeutic agent As described above, when the membrane interacting domain of the propeptides of the present disclosure is stably associated (e.g., conjugated) with one or more therapeutic agents, the propeptides find use in treating conditions associated with FAPα expression (e.g., cancers associated with FAPα expression) in subjects in need thereof.

[0044] Thus, in certain embodiments, a propeptide is provided that includes a membrane-interacting domain stably associated (e.g., conjugated) with one or more therapeutic agents. As used herein, a "therapeutic agent" is a physiologically or pharmacologically active substance capable of producing a desired biological effect at a target site in an animal, such as a mammal or human. A therapeutic agent can be any inorganic compound, organic compound, radioisotope, etc. A therapeutic agent can reduce, inhibit, attenuate, decrease, stop, or stabilize the onset or progression of a disease, disorder, or cell proliferation in an animal, such as a mammal or human. Examples include, but are not limited to, peptides, proteins, nucleic acids (including siRNA, miRNA, and DNA), polymers, small molecules, and radioisotopes. When cell / tissue function is pathological, a therapeutic agent that reduces cell / tissue function can be used. In certain embodiments, the membrane-interacting domain is stably associated with an agent that reduces target cell / tissue function by inhibiting cell proliferation and / or killing the cell / tissue. Such agents may vary and may include radioisotopes, cytostatic agents, and cytotoxic agents, e.g., agents capable of killing target cell tissues whether or not they are internalized within the target cells.

[0045] In certain embodiments, "stably associated" refers to a physical association between two entities in which the average half-life of the association is 1 day or more in PBS at 4°C. In some embodiments, the physical association between the two entities has an average half-life of 1 day or more, 1 week or more, 1 month or more, including 6 months or more, e.g., 1 year or more in PBS at 4°C. According to some embodiments, the stable association results from a covalent bond between the two entities, a non-covalent bond (e.g., an ionic or metallic bond) between the two entities, or other forms of chemical attraction, such as, for example, hydrogen bonding, van der Waals forces, etc. In certain embodiments, the membrane-interacting domain is conjugated to one or more therapeutic agents.

[0046] According to some embodiments, the one or more therapeutic agents comprise a cytotoxic agent, a toxin, a radiosensitizing agent, a radioisotope, or any combination thereof.

[0047] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, maytansine DM1, maytansine DM4, DM-1, an auristatin, or other dolastatin derivatives such as auristatin E or auristatin F, AEB (AEB-071), AEVB (5-benzoylvaleric acid-AE ester), AEFP (antibody-endostatin fusion protein), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepine (PBD), eleutherobin, netropsin, or any combination thereof.

[0048] According to some embodiments, the agent is a toxin, e.g., hemiasterlin and hemiasterlin analogs such as HTI-286 (see, e.g., USPN 7,579,323, WO 2004 / 026293, and USPN 8,129,407, the complete disclosures of which are incorporated herein by reference), abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulinum toxin, shiga toxin, endotoxin, pseudomonas exotoxin, pseudomonas endotoxin, tetanus toxin, pertussis toxin, The protein toxin is selected from anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, aflatoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptin, taicatoxin, calcicludin, geldanamycin, gelonin, lotaustralin, ochratoxin A, patulin, ricin, strychnine, trichothecene, zearlenone, and tetradotoxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0049] In certain embodiments, the agent is a radiosensitizing agent. As used herein, a "radiosensitizing agent" is an agent that enhances the ability of radiation to kill cells (e.g., tumor cells). Non-limiting examples of radiosensitizing agents that can be conjugated to the membrane-interacting domain include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0050] In certain embodiments, the agent is a radioisotope useful, for example, for therapy and / or detection (e.g., imaging). Non-limiting examples of radioisotopes that can be conjugated to the membrane-interacting domain include: 225 Ac, 111 Ag, 114 Ag, 71 As, 72 As, 77 As, 211 At, 198 Au, 199 Au, 212 Bi, 213 Bi, 75 Br, 76 Br, 11 C. 13 C. 55 Co, 62 Cu, 64 Cu, 67 Cu, 165 Dy, 166 Dy, 169 Er, 18 F, 19 F, 52 Fe, 59 Fe, 66 Ga, 67 Ga, 68 Ga, 72 Ga, 154-158 Gd, 157 Gd, 159 Gd, 166 Ho, 120 I, 121 I, 123 I, 124 I, 125 I, 131 I, 110 In, 111 In, 113m In, 194 Ir,81m Kr, 177 Lu, 51 Mn, 52 Mn, 99 Mo, 13 N, 15 N, 15 O. 17 O. 32 P, 33 P, 211 Pb, 212 Pb, 109 Pd, 149 Pm, 151 Pm, 142 Pr, 143 Pr, 191 PT, 193m PT, 195m Pt, 223 Ra, 142 Rb, 186 Re, 188 Re, 189 Re, 105 Rh, 47 Sc, 75 Se, 153 Sm, 117m Sn, 121 Sn, 83 Sr, 89 Sr, 161 Tb, 94 Tc, 99 Tc, 99m Tc, 227 Th, 201 Tl, 172 Tm, 127 Te, 90 Y, 169 Yb, 175 Yb, 133 X, and 89 According to some embodiments, the membrane interaction domain includes, but is not limited to, Zr. 64 Cu (copper-64), 177 Lu (lutetium-177), or 225 It is conjugated to Ac (actinium-225).

[0051] In certain embodiments, the radioisotope is conjugated to the membrane-interacting domain via a chelating agent, non-limiting examples of which include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), MACROPA, and the like.

[0052] Methods for Producing Propeptides The propeptides of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). When polypeptides are chemically synthesized, synthesis can proceed via solution phase or solid phase. Solid-phase synthesis (SPPS) allows the incorporation of unnatural amino acids and peptide / protein backbone modifications. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing the propeptides of the present disclosure. Details of chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. MedChem. 6:3-10, and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8). Briefly, small, insoluble, porous beads are treated with functional units from which peptide chains are constructed. After repeated coupling / deprotection cycles, the free N-terminal amine of the solid-phase-bound peptide or amino acid is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which additional amino acids may be coupled. The peptide remains immobilized on the solid phase and undergoes a filtration process before being cleaved.

[0053] In one non-limiting example, the propeptides of the present disclosure can be synthesized by Fmoc solid-phase synthesis on a Biotage SyroII peptide synthesizer at ambient temperature. The synthesis scale can be 12.5 μM using a preloaded lysine (2-dinitrophenyl) Wang resin, to which a DNP quencher can be linked to the epsilon nitrogen of lysine. The coupling reaction can be carried out with 4.9 equivalents of HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), 5 equivalents of Fmoc-amino acid-OH, and 20 equivalents of N-methylmorpholine (NMM) in 500 μL of N,N-dimethylformamide (DMF) for 8 minutes with shaking. Each amino acid position may be double-coupled, followed by Fmoc deprotection using 500 μL of 40% 4-methylpiperazine in DMF for 10 minutes, followed by six 500 μL DMF washes for 3 minutes. The propeptide may be cleaved from the Wang resin with 500 μL of a solution consisting of 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane for 1 hour with shaking. The crude propeptide product may then be precipitated in 30 mL of cold 1:1 diethyl ether:hexane and then solubilized in a 1:1:1 mixture of DMSO:water:acetonitrile. The solubilized crude product may be purified by high-performance liquid chromatography (HPLC) using an Agilent Pursuit 5 C18 column (5 mm bead size, 150 × 21.2 mm) on an Agilent PrepStar 218 series preparative HPLC. Mobile phases A and B may be water + 0.1% TFA and acetonitrile + 0.1% TFA, respectively. Solvent may be removed under reduced pressure, and the purified peptide product may be solubilized in DMSO stock at the desired final concentration, e.g., 10 mM. Purity may be confirmed by liquid chromatography-mass spectrometry, and stocks may be stored at -20°C.

[0054] When the propeptide is produced using recombinant techniques, it may be produced as an intracellular protein or as a secreted protein using any suitable construct and any suitable host cell, which may be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E. coli) or yeast host cell, respectively.

[0055] Other examples of eukaryotic cells that can be used as host cells include insect cells, mammalian cells, and / or plant cells. When mammalian host cells are used, the cells can include one or more of the following: human cells (e.g., HeLa, 293, H9, and Jurkat cells), mouse cells (e.g., X3, NIH3T3, pancreatic ductal adenocarcinoma 2.1, L cells, and C127 cells), primate cells (e.g., Cos1, Cos7, and CV1), and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

[0056] A wide variety of host-vector systems suitable for expressing the target propeptide can be used according to standard procedures known in the art. See, for example, Sambrook et al. 1989 Current Protocols in Molecular Biology, Cold Spring Harbor Press, New York, and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate method, etc. The transfer method can be selected to provide stable expression of the introduced polypeptide-encoding nucleic acid. The polypeptide-encoding nucleic acid can be provided as an inherited episomal element (e.g., a plasmid) or can be genomically integrated. A variety of suitable vectors are commercially available for use in producing the target polypeptide.

[0057] Vectors can provide for extrachromosomal maintenance in host cells or for integration into the host cell genome. Expression vectors can provide transcriptional and translational regulatory sequences and provide for inducible or constitutive expression, where a coding region is operably linked under the transcriptional control of a transcriptional initiation region and a transcriptional and translational termination region. Generally, transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be strong constitutive promoters (e.g., T7). Expression constructs generally have convenient restriction sites located near the promoter sequence to accommodate insertion of a nucleic acid sequence encoding a protein of interest. A selectable marker acting on the expression host can be present to facilitate selection of cells containing the vector. In addition, expression constructs can contain additional elements. For example, an expression vector can have one or two replication systems, thus enabling it to be maintained in organisms, for example, in mammalian or insect cells for expression and in prokaryotic hosts for cloning and amplification. In addition, the expression construct may contain a selectable marker gene to allow selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.

[0058] Protein isolation and purification can be achieved according to methods known in the art. For example, proteins can be isolated from lysates of cells genetically modified to express the protein constitutively and / or upon induction, or from synthesis reaction mixtures, by immunoaffinity purification, which generally involves contacting the sample with an anti-protein antibody, washing to remove nonspecifically bound material, and eluting the specifically bound protein. Isolated proteins can be further purified by dialysis and other methods commonly used in protein purification methods. In one embodiment, proteins can be isolated using metal chelate chromatography. Proteins of the present disclosure may contain modifications to facilitate isolation.

[0059] The subject propeptides can be prepared in a substantially pure or isolated form (e.g., free from other polypeptides). The propeptide can be present in a composition in which the polypeptide is enriched relative to other components that may be present (e.g., other polypeptides or other host cell components). A purified propeptide can be provided such that the propeptide is present in a composition that is substantially free of other expressed proteins, e.g., a composition in which less than 98%, less than 95%, less than 90%, less than 80%, less than 60%, or less than 50% of the composition is made up of other expressed proteins.

[0060] DOTA-FRIP can be synthesized first using the solid-phase peptide synthesis conditions outlined above, then by triple coupling a resin-bound peptide bearing an N-terminal hexanoic acid with 2 equivalents of dota-NHS, 5 equivalents of HCTU, and 20 equivalents of N,N-diisopropylethylamine (DIPEA) for 12 hours.

[0061] composition Aspects of the present disclosure also include compositions. According to some embodiments, the subject compositions comprise any of the propeptides of the present disclosure, including, but not limited to, any of the propeptides described in the Propeptide Section above and in the Experimental Section below.

[0062] In certain embodiments, the compositions of the present disclosure comprise a propeptide present in a liquid medium. The liquid medium may be an aqueous liquid medium such as water or a buffer solution. One or more additives, such as salts (e.g., NaCl, MgCl, KCl, MgSO), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS)), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelators, etc., may be present in such compositions.

[0063] Aspects of the present disclosure further include pharmaceutical compositions, hi some embodiments, a pharmaceutical composition of the present disclosure comprises a propeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0064] The propeptides can be incorporated into various formulations for therapeutic, diagnostic, or theranostic administration. More specifically, the propeptides can be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable excipients or diluents, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, and aerosols.

[0065] Formulations of propeptides for administration to an individual (e.g., suitable for human administration) are generally sterile and may further contain no detectable pyrogens or other contaminants that would contraindicate administration to a patient according to the selected route of administration.

[0066] In pharmaceutical dosage forms, the propeptides can be administered in the form of their pharmaceutically acceptable salts, or the propeptides can be used alone or in suitable associations, as well as in combination with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0067] For oral preparations, the propeptide may be used alone or in combination with suitable additives, for example, with conventional additives such as lactose, mannitol, corn starch, or potato starch, with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin, with disintegrating agents such as corn starch, potato starch, or sodium carboxymethylcellulose, with lubricants such as talc or magnesium stearate, and, if desired, with diluents, buffers, wetting agents, preservatives, and flavoring agents, to form tablets, powders, granules, or capsules.

[0068] The propeptide can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, intratumoral, etc.) administration. In certain embodiments, the propeptide is formulated for injection by dissolving, suspending, or emulsifying the propeptide in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, along with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed.

[0069] Pharmaceutical compositions containing the propeptide can be prepared by mixing the propeptide having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or tonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, and the like. amino acids such as sucrose, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0070] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and the lyophilized preparation should be reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equal to the volume removed during lyophilization), but solutions containing antimicrobial agents can be used to produce pharmaceutical compositions for parenteral administration.

[0071] Aqueous formulations of the propeptide can be prepared in a pH buffer solution, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively, about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0072] A tonicity agent may be included to adjust the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as physiological salt solution or serum. The tonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0073] Surfactants may also be added to the formulation to reduce aggregation and / or minimize the formation of fine particles and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary surfactant concentrations can range from about 0.001% to about 1% w / v.

[0074] Cryoprotectants may also be added to protect the propeptide against destabilizing conditions during the lyophilization process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Cryoprotectants may be included in an amount of, for example, about 10 mM to 500 nM.

[0075] In some embodiments, the pharmaceutical composition comprises a propeptide and one or more of the above-identified components (e.g., surfactant, buffer, stabilizer, tonicity agent), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, for example, at a concentration ranging from about 0.001 to about 2% (w / v).

[0076] kit Aspects of the present disclosure also include kits. According to some embodiments, the subject kits comprise any of the propeptides of the present disclosure, including, but not limited to, any of the propeptides described in the Propeptides section above and in the Experimental section below.

[0077] In certain embodiments, the kit finds use in practicing the methods of the disclosure, such as methods of treating a condition associated with FAPα expression in a subject in need of such treatment, methods of assessing FAPα activity in a subject, and / or the like.

[0078] Thus, in certain embodiments, kits of the present disclosure may include any of the propeptides or pharmaceutical compositions of the present disclosure and instructions for administering the propeptide or pharmaceutical composition to a subject in need thereof.

[0079] Kits of the present disclosure may include a quantity of a propeptide or pharmaceutical composition present in a unit dose, e.g., ampoule, or multi-dose format. Thus, in certain embodiments, a kit may include one or more (e.g., two or more) unit doses (e.g., ampoules) of a composition comprising a propeptide of the present disclosure. As used herein, the term "unit dose" refers to a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of a composition calculated to be sufficient to produce a desired effect. The amount of a unit dose depends on various factors, such as the particular propeptide used, the effect to be achieved, and the pharmacodynamics associated with the propeptide in the subject. In yet other embodiments, a kit may include a single multi-dose amount of a composition.

[0080] The instructions included in the kit (e.g., instructions for use (IFU)) can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present in the kit as a package insert, on labeling of a container of the kit or its components (i.e., associated with the packaging or subpackaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g., a portable flash drive, DVD, CD-ROM, diskette, etc. In still other embodiments, the actual instructions are not present in the kit, but means are provided for obtaining the instructions from a remote source, e.g., via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. Similar to the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0081] How to use Aspects of the present disclosure include methods of using the propeptides of the present disclosure, which are useful in a variety of contexts, including in vitro and / or in vivo research and / or clinical applications.

[0082] In certain aspects, a method of treating a condition associated with FAPα expression in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a propeptide of the present disclosure.

[0083] In some embodiments, the condition associated with FAPα expression is cancer. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. The subject method can be used to treat a wide variety of cancers. Normal tissues have low and generally undetectable levels of FAPα expression. However, FAPα is overexpressed in many tumor tissues, including breast cancer, colorectal cancer, pancreatic cancer, lung cancer, brain cancer, intrahepatic bile duct cancer, and ovarian cancer. In addition, high levels of FAPα expression can be detected in some tumors derived from non-epithelial tissues, such as melanoma and myeloma. In these tumors, FAPα overexpression is often observed in the stroma, leading to its being considered a universal marker for CAFs, although FAPα can also be detected in gastric cancer, pancreatic cancer, and melanoma cells. Details regarding FAPα expression in human malignancies can be found, for example, in Busek et al. (2018) Frontiers In Bioscience, Landmark 23:1933-1968.

[0084] Thus, in some embodiments, when the condition associated with FAPα expression is cancer, the cancer may be characterized by FAPα expression on cancer cells, cancer-associated fibroblasts, or both. In certain embodiments, the cancer comprises a solid tumor. Examples of solid tumors treatable by the methods of the present disclosure include carcinoma, lymphoma, blastoma, and sarcoma. Non-limiting examples of cancers treatable by the methods of the present disclosure include basal cell carcinoma, squamous cell carcinoma of the skin, oral squamous cell carcinoma, melanoma, esophageal cancer, gastric cancer, colorectal cancer, pancreatic adenocarcinoma, hepatocellular carcinoma, non-small cell lung cancer, mesothelioma, breast cancer, renal cancer, prostate cancer, cervical cancer, ovarian cancer, glioma, parathyroid carcinoma, sarcoma, and myeloma.

[0085] In certain embodiments, when the condition associated with FAPα expression is cancer, the cancer may comprise a hematological malignancy. According to some embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

[0086] Conditions related to FAPα expression other than cancer can also be treated by the method of the present disclosure.For example, the condition related to FAPα expression can be fibrotic disease.In some embodiments, the fibrotic disease comprises liver fibrosis.According to certain embodiments, the fibrotic disease is interstitial lung disease.

[0087] The propeptides of the present disclosure may be administered in compositions in therapeutically effective amounts. By "therapeutically effective amount" is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic (including prophylactic) result, such as a reduction in symptoms of cancer, fibrotic disease, or other conditions associated with FAPα expression, compared to a control. With respect to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow tumor growth, reduce tumor size, and / or the like. An effective amount can be administered in one or more administrations.

[0088] As noted above, aspects of the present disclosure include methods for treating a condition associated with FAPα expression in a subject in need thereof. "Treatment" or "treating" refers to at least the amelioration of one or more symptoms associated with the condition associated with FAPα expression (e.g., cancer, fibrotic disease, etc.), where amelioration is used broadly to refer to at least a reduction in a parameter associated with the condition being treated, e.g., the magnitude of the symptom. Thus, treatment also includes situations in which a condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0089] The propeptides of the present disclosure may be administered to a subject alone or in combination with a second agent of interest, including, but not limited to, an agent approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use in the treatment of conditions associated with FAPα expression, such as cancer, fibrotic diseases, and the like.

[0090] The present disclosure also provides a method for detectably labeling a phospholipid bilayer of a cell in the presence of FAPα activity. Such a method comprises contacting a propeptide of the present disclosure with a FAPα that contributes to FAPα activity. The membrane-interacting domain is detectably labeled, and the FAPα cleavage site of the propeptide is cleaved by FAPα to release a cleavage product containing the detectably labeled membrane-interacting domain, such that the detectably labeled membrane-interacting domain interacts with the phospholipid bilayer of the cell and detectably labels the phospholipid bilayer of the cell in the presence of FAPα activity.

[0091] Aspects of the present disclosure further include methods of assessing FAPα activity in a subject. Such methods include administering to a subject a propeptide of the present disclosure, wherein the membrane-interacting domain is detectably labeled, and at a site of FAPα activity in the subject, the FAPα cleavage site of the propeptide is cleaved by FAPα to release a cleavage product comprising the detectably labeled membrane-interacting domain, which cleavage product interacts with the phospholipid bilayer of a cell at the site of FAPα activity in the subject. Such methods further include assessing for the presence or absence of cells labeled with the cleavage product, wherein the presence of cells labeled with the cleavage product indicates FAPα activity in the subject.

[0092] In certain embodiments, the membrane interaction domain is detectably labeled with a labeling agent that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, single-photon emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, etc. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, etc. In certain aspects, the labeling agent is a multimodal in vivo imaging agent that allows in vivo imaging using two or more imaging techniques (see, for example, Thorp-Greenwood and Coogan (2011) Dalton Trans. 40: 6129-6143).

[0093] In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in near-infrared (NIR) imaging applications. Such agents include, but are not limited to, Kodak X-SIGHT dyes, Pz247, DyLight 750 and 800 Fluor, Cy5.5 and 7 Fluor, Alexa Fluor 680 and 750 dyes, IRDye 680 and 800CW Fluor. According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, and non-limiting examples of in vivo imaging agents include: 99m Tc, 111 In, 123 I, 201 Tl, and 133 In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, e.g., 11 C. 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 Ga, 177 Lu, 225 Ac, etc.

[0094] As will be appreciated with the benefit of this disclosure, the propeptides of the present disclosure find use in theranostic applications. By way of example, the membrane-interacting domain can be coupled to a radioisotope (e.g., 64 Cu (copper-64), 177 Lu (lutetium-177), or 225 When labeled with Ac (actinium-225), the propeptide may be used in a method of treating a condition associated with FAPα expression (e.g., cancer, fibrotic disease, etc.) in a subject in need of such treatment, wherein the method further comprises assessing FAPα activity in the subject using a radioisotope for in vivo imaging, e.g., by PET / CT and / or PET / MR.

[0095] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0096] experiment Example 1 - Identification of unique FAPα substrate sequences using multiplex substrate profiling by mass spectrometry (MSP-MS) Although much is known about the substrate preferences for FAPα from P4-P1, the optimal P4-P4' substrate sequence for FAPα has not previously been defined. In this example, multiplex substrate profiling by mass spectrometry (MSP-MS) was performed to identify candidate FAPα P4-P4' substrate sequences that could be included in FAPα target-restricted interacting peptides (or "FRIPs").

[0097] MSP-MS yielded several potential substrates, nearly all of which possessed Gly-Pro at positions P2-P1, previously shown to be essential for FAPα proteolysis. The top of Figure 3 shows an Icelogo representing cumulative amino acid preferences for recombinant human FAPα. The bottom of Figure 3 shows a heatmap summarizing the most frequently incorporated amino acids at each site in the cleavage products. Brighter indicates enriched amino acids. X = no preferred amino acid.

[0098] A list of the top P4-P4' peptides cleaved by human FAP alpha, as determined using MSP-MS substrate profiling technology, is shown in the table below, where X indicates an undefined amino acid site. [Table 2]

[0099] Three top hits were selected for further analysis: HIGPTAAY (SEQ ID NO: 38), HQGPFWML (SEQ ID NO: 40), and HYGPTVNK (SEQ ID NO: 42). 8-mer peptides carrying FRET donor-acceptor dyes on the N- and C-termini were synthesized and evaluated as substrates for recombinant human FAPa (RND Systems) under Michaelis-Menten conditions. The kcat / Km were approximately 120,000, 80,000, and 15,000 M, respectively. -1 seconds -1 It was decided that:

[0100] Provided below is a table summarizing the kinetic properties of three of the top P4-P4' 8mer peptides emerging from the MSP-MS assay, which were determined using FRET donor / acceptor substrate adducts and human recombinant FAP alpha. [Table 3]

[0101] Data showing the kinetic properties for probe 1, probe 2, and probe 3 are provided in Figures 4A, 4B, and 4C, respectively.

[0102] Example 2 - Development of FAPα-targeted restrictive interacting peptides (FRIPs) and selective uptake in FAPα-positive tumors in vivo In this example, the three top truncation sequences from the previous examples were incorporated into full-length RIPs bearing an N-terminal DOTA for radiolabeling (referred to herein as "FRIP1," "FRIP2," and "FRIP3"). A schematic diagram of an example FRIP with the probe 2 sequence is provided at the top of Figure 5. FRIPs are: 64The target gene includes (1) a Cu-labeled membrane-interacting / binding domain, (2) a FAPα cleavage site, and (3) a masking domain. In this example, the membrane-interacting domain is an antimicrobial peptide (AMP), specifically, the temporin L peptide (FVQWFSKFLGK, SEQ ID NO: 51) as the AMP. The FAPα cleavage site is the top hit identified in Example 1. In this example, the masking domain is a peptide derived from protease-activated receptor 1 (PAR1), which has the sequence QDPNDQYEPF (SEQ ID NO: 35).

[0103] DOTA-FRIP was radiolabeled with Cu-64 using the protocol developed for DOTA-GRIP B (Zhao et al. (2021) ACS Cent Sci. 7(10):1638-49). Radiochemical yields were consistently >95% and radiochemical purities >99%. 64 The cleavage of Cu-FRIP was examined in vitro by HPLC. 64 Cu-FRIP2 was cleaved most efficiently, with complete conversion to a product peak within 3 hours. 64 The in vitro serum stability of Cu-FRIP2 was tested and found to be >95% stable for up to 12 hours at room temperature.

[0104] Next, in mice bearing U87 xenografts 64 Cu-FRIP2 uptake, 64 Cu-FRIP1, 64 Cu-FRIP3, and 64 Compared to Cu-GRIP B. 24 hours after injection, 64 Tumor uptake of Cu-FRIP2 was significantly higher than that of the other probes (Figures 6A-6B), highlighting that efficient proteolytic cleavage is required for tumor retention of RIPs.

[0105] next, 64 We tested whether Cu-FRIP2 is selectively taken up by FAPα-positive tumors in vivo. 64Cu-FRIP2 (approximately 400 mCi / mouse, n=4 per arm) was administered to different cohorts of mice bearing subcutaneous U87 MG (FAPα-positive human glioblastoma), HT1080 (FAPα-negative human fibrosarcoma), or MIA Paca-2 tumors (FAPa-negative human pancreatic cancer). 24 hours after injection, 64 Cu-FRIP2 uptake was significantly higher in U87 tumors compared to models lacking FAPα (Figure 7). Biodistribution data collected 24 hours after injection (Figure 8) showed that U87 tumors had the highest uptake of the radiotracer compared to normal tissues. The liver and kidney also showed moderate uptake of the radiotracer. Similar to GRIP B, uptake in normal mouse tissues was generally low. The molecule was excreted through the kidney, with some retention in the liver.

[0106] in male and female C57Bl6 mice 64 Formal dosimetry studies of Cu-FRIP2 have shown that the effective systemic dose of the tracer is 64 Cu-GRIP B and 18 The highest dose was shown to be within the range of other commonly used tracers such as F-FDG. 64 As documented for Cu-GRIP B, it was experienced by the liver and kidneys.

[0107] Example 3 - FRIP shows superior biodistribution compared to FAPI 46 In this example, a longitudinal PET study was performed to 64 Tumor uptake of Cu-FRIP2 was compared to FAPI-46, an active site-directed FAPα radioligand currently in clinical trials (Meyer et al. (2020) J Nucl Med. 61(8):1171-7). DOTA-FAPI-46 was purchased from MedChemExpress. 64 A similar protocol for Cu-FRIP2 was used for coupling to Cu-64, with radiochemical yields and purities >95%.

[0108] 64 Cu-FRIP2 and 64 Cu-FAPI 46 was administered at equal doses and specific activity to mice bearing subcutaneous U87 tumors. PET studies (Figure 9A) showed that 64 Tumor uptake of Cu-FRIP increased from 2 to 24 hours after injection, with an SUV of approximately 9% ID / cc. 平均 (Figure 9B). 64 Cu-FAPI 46 levels were significantly lower (SUV 平均 Approximately 1% ID / cc). In particular, 64 Tumor uptake of Cu-FRIP2 was 64 Although significantly higher than Cu-FAPI 46, the clearance rates of each radiopharmaceutical were virtually identical. 64 Cu-FRIP2 and 64 Tumor uptake of Cu-FAPI 46 was evaluated in a separate cohort of mice bearing PC3 PIP xenografts, which have a lower FAP compared to U87. PET data (Figures 10A-10B) showed that Cu-FAPI 46 was significantly increased in the tumor. 64 Cu-FRIP2 uptake 64 The results showed that the IFN-γ-FAP1 / ... 64 The area under the tumor curve for Cu-FRIP2 is 64 It was about five times higher than Cu-FAPI 46. 64 Cu-FRIP2 uptake was significantly lower in PC3 PIP tumors compared to U87 tumors, as expected. [Table 4]

[0109] Based on mouse dosimetry studies 64 The estimated human absorbed dose for Cu-FRIP2 is provided in the table below, values ​​were calculated using OLINDA EXM software. [Table 5]

[0110] Example 4 67 Cu-FRIP2 potently suppresses tumor growth Based on the data provided in the preceding examples herein, it was predicted that RIPs (including FRIPs) would be advantageous for targeted radiation therapy (TRT). In contrast to RLTs, RIPs accumulate in tumors through a renewable catalytic mechanism, but also persist within tumors because cancer cells lack efficient mechanisms for degrading peptide / membrane complexes (FIG. 11). In this example, we present a non-limiting example of a FRIP ( 67 Proof-of-concept data using Cu-FRIP2) are provided.

[0111] Antitumor evaluation studies were performed in mice bearing U87 MG tumors. 67 Antitumor evaluation studies were conducted using Cu-67 because it is a beta-emitter and is isostere to Cu-64. 67 It is of clinical interest, as demonstrated by a recent trial in the United States targeting SSTR2-positive neuroendocrine tumors with Cu-SARTATE (NCT04023331). 67 To synthesize Cu-FRIP2, 64 Using the same protocol applied to Cu-FRIP2, 67 CuCl2 was coupled to DOTA-FRIP2. The radiotracer was synthesized to a radiochemical yield and purity of >95%.

[0112] Male athymic nu / nu mice bearing subcutaneous U87 MG tumors 67 A single IV dose of Cu-FRIP2 (1 mCi / mouse) or vehicle (n=8 mice / arm) was given. Data are shown in Figures 12A-12C, 13A-13B, and 14. 67 A single dose of Cu-FRIP2 significantly inhibited U87 MG tumor growth (Figure 12A) and increased survival (Figure 13A). 3The endpoints of (1) tumor volume greater than 100% or (2) weight loss greater than 20% were used as benchmarks. Furthermore, treatment was well tolerated, as no significant reduction in body mass was detected (Figure 13B). Spider plots showing the change in individual tumor volume for the mice represented are shown in Figure 14.

[0113] next, 67 Cu-FRIP2 67 We examined whether IFN-γ-FAPI 46 inhibited tumor growth more potently than Cu-FAPI 46. The data are shown in Figures 15A-15B and 16. Mice bearing subcutaneous U87 MG xenografts were treated with either vehicle or 67 Cu-FRIP2 (1 mCi / mouse) or 67 Treatment was with a single IV dose of Cu-FAPI 46 (1 mCi / mouse). 67 Cu-FRIP2 is 67 Compared with Cu-FAPI 46 or vehicle, it more potently inhibited tumor growth.

[0114] Then, it is given in high doses 64 We evaluated whether Cu-FRIP2 can suppress tumor growth. Male athymic nu / nu mice bearing subcutaneous U87 MG xenografts were treated with vehicle or 64 and treated with an IV dose of Cu-FRIP2 (1.5 mCi / mouse) (n=8 mice / arm). 67 Cu-FRIP2 inhibited tumor growth compared to vehicle (Figure 19A). Survival data plotted on Kaplan-Meier curves showed significantly prolonged survival (P < 0.01) in the treated arm compared to the vehicle arm (Figure 19B).

[0115] Accordingly, the foregoing merely illustrates the principles of the present disclosure. Those skilled in the art will recognize that, although not explicitly described or shown herein, they can devise various configurations that embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the art, and should be construed without limitation to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed that perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. A propeptide, Membrane interaction domain, A masking domain that, when linked to the membrane interaction domain, is effective in inhibiting the interaction of the membrane interaction domain with the phospholipid bilayer, A propeptide comprising a fibroblast-activating protein alpha (FAPα) cleavage site positioned between the membrane interaction domain and the masking domain.

2. The propeptide according to claim 1, wherein the membrane interaction domain comprises a protein from the temporin family.

3. Said membrane interaction domain comprises the amino acid sequence X ａ X ｂ X ｃ X ｄ X ｅ X ｆ Y ａ X ｇ X ｈ Y ｂ Y ＊ X ｉ X ｊ , wherein in the formula, X ａ , X ｂ , X ｄ , X ｅ , X ｇ , X ｈ , X ｉ and X ｊ are hydrophobic amino acid residues, Y ａ and Y ｂ are hydrophilic amino acid residues, and Y ＊ is a charged amino acid residue. The propeptide according to claim 2.

4. The propeptide according to claim 3, wherein the membrane interaction domain comprises the amino acid sequence FVQWFSKFLGKLL (SEQ ID NO: 3) or the amino acid sequence FVQWFSKFLGRIL (SEQ ID NO: 2).

5. The propeptide according to claim 1, wherein the masking domain comprises a peptide derived from protease-activated receptor-1 (PAR-1).

6. The masking domain is an amino acid sequence (X ａ ) (X ｂ )PND(X ｃ ) Includes YEPF (Sequence ID 52), in the formula, X ａ However, R or Q, and X ｂ However, it is N or D, and X ｃ The propeptide according to claim 5, wherein the propeptide is K or Q.

7. The propeptide according to claim 6, wherein the masking domain comprises the amino acid sequence QDPNDQYEPF (SEQ ID NO: 35) or the amino acid sequence RNPNDKYEPF (SEQ ID NO: 36).

8. The propeptide according to claim 1, wherein the FAPα cleavage site contains the FAPα-specific motif GP at the P2-P1 position.

9. The propeptide according to claim 8, wherein the FAPα cleavage site comprises the amino acid sequence YHGPLAHX (SEQ ID NO: 37), HIGPTAAY (SEQ ID NO: 38), XXIPTNIR (SEQ ID NO: 39), HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), HYGPTVNK (SEQ ID NO: 42), XXWPMGMY (SEQ ID NO: 43), XXFPNMWS (SEQ ID NO: 44), XXGSQVFS (SEQ ID NO: 45), XXMPEEVA (SEQ ID NO: 46), or XXHPTKSF (SEQ ID NO: 47), where X is any amino acid.

10. The propeptide according to claim 1, comprising the membrane interaction domain, the FAPα cleavage site, and the masking domain from the N-terminus to the C-terminus.

11. The propeptide according to claim 1, wherein the membrane interaction domain is conjugated to one or more therapeutic agents.

12. The propeptide according to claim 11, wherein the one or more therapeutic agents include a cytotoxic agent, a toxin, a radiosensitizing agent, a radioisotope, or any combination thereof.

13. The one or more therapeutic agents described above contain a radioactive isotope, and the radioactive isotope is (a) Actinium-225, Astatine-211, Bismuth-212, Bismuth-213, Bromine-76, Bromine-77, Calcium-47, Carbon-11, Carbon-14, Chromium-51, Cobalt-57, Cobalt-58, Copper-64, Erbium-169, Fluorine-18, Gallium-67, Gallium-68, Hydrogen-3, Indium-111, Iodine-123, Iodine-125, Iodine-131, Iron-59, C Selected from the group consisting of Lipton-81m, Lead-212, Lutetium-177, Nitrogen-13, Oxygen-15, Phosphorus-32, Radium-223, Radium-224, Samarium-153, Selenium-75, Sodium-22, Sodium-24, Strontium-89, Technetium-99m, Thallium-201, Thorium-226, Thorium-227, Xenon-133, or Yttrium-9, and / or (b) Conjugated to the membrane interaction domain via the chelated portion, The propeptide according to claim 12.

14. The propeptide according to claim 13, wherein the chelated portion comprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

15. A pharmaceutical composition, A propeptide according to any one of claims 1 to 14, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a propeptide according to any one of claims 11 to 14 for use in a method of treating a subject requiring treatment for a condition related to FAPα expression, wherein the method comprises administering an effective amount of the propeptide to the subject.

17. The state related to FAPα expression is, (a) having cancer, or (b) It is a fibrous disease, The pharmaceutical composition according to claim 16.

18. The aforementioned cancer, (a) A cancer characterized by FAPα expression on cancer cells, cancer-associated fibroblasts, or both: (b) including and / or solid tumors (c) It is a carcinoma, lymphoma, blastoma, or sarcoma. The pharmaceutical composition according to claim 17.

19. The pharmaceutical composition according to claim 17, wherein the cancer is basal cell carcinoma, squamous cell carcinoma of the skin, squamous cell carcinoma of the oral cavity, melanoma, esophageal cancer, gastric cancer, colorectal cancer, pancreatic adenocarcinoma, hepatocellular carcinoma, non-small cell lung cancer, mesothelioma, breast cancer, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, glioma, parathyroid cancer, sarcoma, or myeloma.

20. The pharmaceutical composition according to claim 17, wherein the cancer includes hematological malignancies.

21. The pharmaceutical composition according to claim 20, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

22. The pharmaceutical composition according to claim 16, wherein the membrane interaction domain includes a detectable label, and the method further comprises performing in vivo imaging on cells in the subject labeled with the membrane interaction domain.

23. (a) The one or more therapeutic agents include the detectable label and / or (b) The in vivo imaging includes in vivo positron emission tomography (PET), The method according to claim 22.

24. A method for detecting the phospholipid bilayer of cells in the presence of FAPα activity, The method involves contacting the propeptide according to any one of claims 1 to 14 with FAPα that contributes to the FAPα activity, The aforementioned membrane interaction domain is labeled in a detectable manner. A method comprising the following steps: the FAPα cleavage site of the propeptide is cleaved by FAPα, releasing a cleavage product containing a detectably labeled membrane interaction domain, the detectably labeled membrane interaction domain interacting with the phospholipid bilayer of the cell, and detectingly labeling the phospholipid bilayer of the cell in the presence of FAPα activity.

25. A method for evaluating FAPα activity in a subject, The process includes evaluating the presence or absence of cells labeled with the cleavage product in a subject who has been administered the propeptide according to any one of claims 1 to 14, The membrane interaction domain is detectably labeled, and at the FAPα activity site in the subject, the FAPα cleavage site of the propeptide is cleaved by FAPα, releasing the cleavage product containing the detectably labeled membrane interaction domain, and the cleavage product interacts with the cellular phospholipid bilayer at the FAPα activity site in the subject. A method wherein the presence of cells labeled with the cleavage product indicates FAPα activity in the subject.

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